Inorganic column packings such as silica and alumina have been used for years for separation of a wide variety of basic neutral and acidic compounds. During the past few years increased interest in the separation of polar biological compounds which are sensitive to both pH and solvent has led to a great deal of work to modify the polar hydroxyl groups on inorganic packing materials. These modifications are necessary to prevent degradation of either the biological compound or the packing material.
Nucleic acid constituents have been separated by ion exchange chromatography using strong anion or cation exchange functions on cross-linked polystyrenedivinylbenzene resins. Exemplary is Kratovich and Roe, Journal of Chromatography, Vol. 155, 407 (1978), which describes the use of a polystyrene-divinylbenzene 8% cross-linked quaternary amine strong anion exchange column to achieve baseline separation of the 2'- and 3'-isomers of AMP and GMP and partial resolution of the 2'- and 3'-isomers of CMP and UMP.
It is known to modify a siliceous surface by reaction with monohalogenated silanes, silazanes or silylamines, or with monoalkyl or monoacetoxysilanes and to chemically modify the reaction product. This type of prior art is illustrated by U.S. Pat. No. 4,043,905 to Novotny et al. Similarly, modification of silica by reaction with benzyltrichlorosilane, followed by modification of the product to a benzylethylhydroxyl dimethyl ammonium ion is known. This type of prior art is exemplified by Unger and Nyamah, Chromatographia, Vol. 7, No. 2, 63-68 (1974). Also, it is known to form a silica having a quaternary ammonium ion bonded thereto by reacting chlorodimethyl [4-(4-chloromethylphenyl)butyl] silane with silica, followed by reaction with trimethylamine. This procedure produces a strong anion exchange composition, with a capacity of approximately 200 microequivalents/gram which only weakly retains and resolves some of the purine nucleotides. This type of art is exemplified by Asmus et al, Journal of Chromatography, Vol. 119, 25-32 (1976).
Furthermore, a silica modified with 3-chloropropyltrichlorosilane followed by triethylamine is known, as illustrated by Cox et al, Journal of Chromatography, Vol. 117, 269-278 (1976). However, this modified silica has a very low anion exchange capacity. The silica-silane reaction is carried out under reflux in dioxane.
Moreover, a proprietary strong anion exchange composition having a trimethylammonium ion bonded to a silica by an undisclosed linking group is known. This modified silica, known as Partisil-10 SAX (Whatman), is offered as a pellicular packing (37-50.mu.) and has very low capacity (10-15 .mu.eg/g). In Whatman 1976, Bulletin 116, "Analysis of Nucleic Acid Constituents by High Performance Liquid Chromatography", the chromatogram at page 5, entitled "2', 3' Mononucleotides on Partisil-10-SAX", shows that this material produces only a partial resolution of the 2'- and 3'-monophosphate nucleotides of adenine, uracil, guanine, and cytosine.
Additionally, the reaction of silica with a substituted silane in water is known, as shown by Majors, Journal of Chromatographic Science, Vol. 12, 767 (1974). However, as described in FIG. 1 of this publication, a polymer is formed. The work of C. J. Bossart in ISA Transactions, Vol. 7, 283 (1968) appears to similarly form a polymer by the reaction in water at reflux of silica and a C.sub.6 -trichlorosilane. Thus, the prior art of which I am aware has not produced a strong anion exchange composition that is capable of achieving baseline resolution of 2'- and 3'-isomers of monophosphate nucleotides such as UMP and, furthermore, has not produced a strong anion exchange composition having this capability that also has high capacity. Additionally, this prior art fails to provide a process for forming a strong anion exchange composition in water, without polymer formation, from silica and a silane.
My copending applications, Ser. No. 953,380, filed Oct. 23, 1978, and Ser. No. 16,847, filed Mar. 2, 1979, now U.S. Pat. No. 4,290,892, describe the use of an aqueous solvent in reacting an amine-containing alkoxysilane such as N-2-aminoethyl-3-aminopropyltrimethoxysilane with a silica.